CN1804625B - Method for monitoring molecule conformation transition - Google Patents
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Abstract
The invention discloses a method for monitoring molecule conformational transition. It absorbs the molecule on the single frank surface of the micro cantilever beam and tests the displacement change of the micro cantilever beam leaded by the molecule, if the displacement changes, the molecule conformational transits.
Description
Technical field
The present invention relates to a kind of method of monitoring molecule conformation transition.
Background technology
Big molecule comprises various synthetic high polymers (as tygon) and natural polymer (as DNA, protein, starch etc.).Exist many singly-bounds in the macromolecular main chain.Because rotation in the singly-bound, big molecule can present different spatial shape, i.e. macromolecular conformation.When environment changed, corresponding change (also being called big molecular folding) also can take place in macromolecular conformation thereupon.For example, to the temperature sensitive type macromolecule, heat up or its conformation of cooling meeting change; For protein molecule, temperature, pressure, the variation of pH value etc. all can make its conformational change and cause it to lose original physiologically active.Macromolecular conformation transition is a major issue in the polymer Neo-Confucianism, also is one of basic problem in the life science.
Nineteen sixty Stockmayer (W H Stockmayer.Problems of the statisticalthermodynamics of dilute polymer solutions[J] .Makromol.Chem., 1960,35:54-74) prophesy theoretically first:, have conformation transition from random coil to the bead of collapsing to the big molecule of linear homopolymerization relatively simple for structure (the long-chain shape molecule that forms by identical monomer polymerization).After this, people have carried out a large amount of research.People (Chi Wu such as nineteen ninety-five Wu chi, Shuiqin Zhou.Laser LightScattering Study of the Phase Transi-tion of Poly (N-isopropylacrylamide) in Water.1.Single Chain[J] .Macromolecules 1995,28:8381-8387) utilization laser light scattering method (LLS) research poly-N-isopropyl allylamine (Poly (N-isopropyl acrylamide), PNIPAM) conformation transition of molecule (Fig. 1) in aqueous solution, for the first time from experimentally having observed the thermodynamically stable bead conformation (Fig. 2) of subsiding, illustrated in free solution, the homopolymer macromolecular chain is in the conformation transition process of a ball of string-bead and bead-ball of string, exist four different conformational states, promptly, random coil, a shrinkage ball of string, molten drop and bead (the Chi Wu that subsides fully, Xiaohui Wang.Globule-to-coilTransition of a single homopoly chain in solution.[J] Phy.Rev.Lett.1998,80,4092-4094).In recent years, Many researchers is adsorbed onto solid surface with the macromolecular end of PNIPAM and forms polymer brush (de Gennes, P.G.Conformations of Polymers Attached to anInterface[J] .Macromolecules, 1980.13.1069-1075), utilization QCM (quartz crystal oscillator balance) (Guangming Liu, Guangzhao Zhang.Collapse and swelling of thermallysensitive poly (N-isopropylacryl amide) brushes monitored with a quartzcrystal microbalance[J] .J.Phys.Chem.B 2005,109:743-747), SPR (surface plasma resonance) (Balamurugan S, Mendez S.et al Thermal Response ofPoly (N-isopropyl acrylamide) Brushes Probed by Surface Plasmon Resonance[J] .Langmuir, 2003,19:2545-2549), AFM (atomic force microscope) (AFM Investigationsof Poly (N-isopropylacryl amide) Brushes Prepared by Surface-InitiatedPolymerizations.[J] Adv.Mater.2002,14,1130-1134) etc. method is studied macromolecular conformation transition.Result of study shows, polymer brush subside with free solution in strand subside differently, the conformation transition of polymer brush recurs in than wider temperature range (20 ℃-40 ℃).
The protein conformation folding problem is bio-science one of unsolved significant problem still, also be current active research the most field. the chain biomacromolecule that protein is made up of amino acid. form peptide bond by shrinking between amino acid and the amino acid, and then forming so-called polypeptied chain. polypeptied chain represents to be the primary structure of protein by amino acid. secondary structure then means some basic three-dimensional structures of protein, as alpha-helix, β-sheet, β hair fastener etc. the 3-d modelling that tertiary structure then is made up of some secondary structures, it has reflected concrete configuration and the coordination of polypeptied chain each several part in three dimensions, being the foldable structure of polypeptied chain and having biological function. the formation of quaternary structure then is because the stability of protein and the needs of function in the biosome, is made up of many polypeptied chains usually and forms foldable structure. protein conformation folds and comprises two aspects: 1) protein of sex change or polypeptied chain folds; 2) the amino acid sequence chain of translating into by coding triplet (new polypeptide chain) is folding. and enzyme is that living cells produces and has a class specific proteins of catalytic action, various chemical reactions in the biosome nearly all are by enzymatic. the conformation problem of studying it has great significance.
The monitoring method of protein folding has nuclear magnetic resonance method, circular dichroism method, laser Raman spectrometry, fluorescent spectrometry, ultraviolet spectroscopy, tracer method etc. at present.Wherein nuclear magnetic resonance method can be measured the three-dimensional structure of protein, but it only limits to analysis length and is no more than 150 amino acid residue small proteins.Additive method can be measured the local conformation of protein molecule, but is difficult to obtain the complete three-dimensional structure of protein molecule, has limitation on using.Conformation research with lysozyme is example, studied it at urea and the guanidine hydrochloride conformation folding process during as denaturant with fluorescent spectrometry, confirmed that it is transformed into nature conformation (the ellipsoid shape conformation when being in the phosphate solution) and middle transition process in the influence (urea and guanidine hydrochloride induce the fluorescence phasor method of lysozyme unfolding to study chemical journal 2,003 61.6 803～807) that has or not under the reductive agent from non-activity conformation (being in the loose ball of string conformation that the hydrochloric acid croak solution of 6M or the urea solution of 8M is).
Micro-cantilever sensing experiment technology is to detect the diastrophic a kind of highly sensitive experimental technique that produces on the micro-cantilever by optical lever method (optical beam technique).It uses widely in atomic force microscope (AFM), may detect the displacement of the inferior nanometer scale of little beam end.As can see from Figure 3, the laser beam sent of semiconductor laser focuses on the free end of micro-cantilever.Folded light beam enters the detection target surface of photoelectric position sensor (PSD).When little beam occured bending and deformation, laser beam is deflection thereupon, and this signal of PSD perception also is converted into corresponding electric signal output, and this can realize the detection to the beam distortion.The thermoelectric refrigerating unit control solution temperature of container bottom.
Summary of the invention
The method that the purpose of this invention is to provide a kind of monitoring molecule conformation transition.
The method of a kind of monitoring molecule conformation transition provided by the present invention, be with molecular adsorption (physics or chemisorption) on the micro-cantilever single side surface, the micro-cantilever change in displacement that detection is caused by molecule, the micro-cantilever displacement that is caused by molecule changes as described, and then the conformation of described molecule changes.
The described micro-cantilever displacement that is caused by molecule conformation transition is: causing under the identical environmental baseline of conformation transition that the micro-cantilever displacement of having adsorbed described molecule is poor with the micro-cantilever displacement of not adsorbing described molecule.
Described environmental baseline comprises temperature, pressure, irradiate light, adds physical factor and chemical factors such as organic solvent, urea, guanidine and pH value, ion concentration such as electromagnetic field change.
The thickness of described micro-cantilever can be 0.1-2 μ m, and length can be 40-1000 μ m, and width is more than or equal to 20 μ m.
The material of making micro-cantilever can be SiNx, SiO
2Deng.
Described molecule comprises various synthetic macromolecules (as tygon) and natural macromolecular (as DNA, protein, starch etc.).
Described synthetic macromolecule specifically can be poly-N-isopropyl acrylamide.Described natural macromolecular specifically can be lysozyme.
The present invention is adsorbed onto the PNIPAM strand of thermal sensitivity on the micro-cantilever single side surface by the method for molecule self assembly, when utilizing laser triangulation detection PNIPAM molecular water solution temperature between 20 ℃-40 ℃, to change, by the caused little deflection of beam distortion of the conformation transition of PNIPAM strand; Obtain the information that macromolecular conformation changes by the distortion of little beam. experimental result shows: in temperature-rise period because the transformation (Fig. 2) from extended conformation to the conformation of collapsing has taken place in the PNIPAM strand, cause the surface stress of little beam that variation has taken place, make little beam produce flexural deformation (Fig. 3). reciprocal flexural deformation takes place in little beam in temperature-fall period, corresponding PNIPAM strand returns to the process of extended conformation from the conformation of collapsing. with LLS (detecting the change in radius of big molecular coil), QCM (detection additional mass) is different with SPR detection techniques such as (detecting the thickness refractive index of unimolecular film), what this method detected is intermolecular interaction, detectable molecular weight ranges very wide (10
2～10
7G/mol).The present invention also arrives the micro-cantilever single side surface with the lysozyme molecular adsorption, when detecting at solvent property and change (from phosphate solution PBS to hydrochloric acid croak solution), because the flexural deformation (Figure 10) that the conformation of lysozyme produces from natural folded state (ellipsoid shape conformation) to unfolding attitude (a loose ball of string) micro-cantilever that conformation change caused.
The present invention is based on the mechanical signal in the molecular detection conformation transition process, creatively utilize micro-cantilever sensing experiment technology for detection molecule conformation transition, provided a new visual angle observing the molecule conformation transition process.The present invention is significant for the conformation transition of recognizing molecule from microcosmic.
Description of drawings
Fig. 1 is the PNIPAM molecular structure
Fig. 2 is the conformation transition synoptic diagram of PNIPAM molecule
Fig. 3 is a micro-cantilever sensing experiment system schematic
Fig. 4 causes little beam distortion synoptic diagram for the conformation change of PNIPAM molecule
Fig. 5 is not for adsorbing the deformation curve of little beam of PNIPAM with temperature
Fig. 6 is adsorbed with the deformation curve of little beam of PNIPAM with temperature
Fig. 7 is for only changing caused little beam deformation curve by the folded conformation of PNIPAM molecule with variation of temperature
Little beam surface stress that Fig. 8 causes for the folded conformation of PNIPAM molecule changes is with the variation of temperature curve
Fig. 9 is a PNIPAM conformation transition process synoptic diagram
Figure 10 causes little beam distortion synoptic diagram for the conformation change of lysozyme
Figure 11 is little beam distortion--the time lab diagram of lysozyme
Embodiment
Experimental technique among the following embodiment if no special instructions, is conventional method.
Embodiment 1: utilize micro-cantilever sensing experiment technology for detection PNIPAM molecule conformation transition
Experimental system as shown in Figure 3.The laser beam that semiconductor laser sends focuses on the free end of micro-cantilever.Folded light beam enters the detection target surface of photoelectric position sensor (PSD).Little beam is fixed in the small container of the stainless steel wall that glass cover binds (1ml), little beam length 200um, wide 20um, thick 1um.The solution turnover is by the control of micrometeor peristaltic pump.By the electrothermal refrigeration device control solution temperature of container bottom, its temperature stability is ± 0.01K 18 ℃-42 ℃ of temperature control scopes.When little beam occured bending and deformation, laser beam is deflection thereupon, and PSD is converted into corresponding electric signal output with this signal, can realize the detection to the beam distortion thus.The minimum distinguishable displacement of little beam free end is 0.1nm in the experimental system.
In order to observe the transition phenomenon of pNIPAM molecular conformation with temperature.Need modify with PNIPAM molecule (Fig. 4) on the single side surface of little beam, its process is as follows: at first, and at SiN
xThe Au of evaporation 20～50 nanometer thickness on one side surface of little beam, utilize combining of sulfydryl and Au then, one end is adsorbed with sulfydryl, and (PNIPAM molecular adsorption HS) is to the gold-plated side of little beam, and finally form molecular brush (Milner by self-assemble (self assembly) process, S.T.Science 1991,251,905).The PNIPAM molecular weight that uses in the present embodiment is M
w=1.5 * 10
5G/mol, molecular weight distribution M
w/ M
n=1.16.
Change between 20 ℃ to 40 ℃ by radiator valve control solution temperature, and monitoring distortion of little beam in this process just can obtain the conformation transition information of PNIPAM molecule in this temperature range.The temperature variation mode is as follows: with 1 ℃ be the interval, heat up until 40 ℃ since 20 ℃.Control temperature afterwards in the same way and be cooled to 20 ℃ from 40 ℃.Because two kinds of material coefficient of thermal expansion coefficients of SiNx and Au are different, little beam self also can produce distortion during temperature variation---two material temperature change effects.In order only to obtain change information, before absorption PNIPAM molecule and carried out the circulation experiment of twice temperature afterwards respectively by the PNIPAM molecule.After subtracting each other, both just can remove two material temperature change effects.Be bent into the forward of distortion in the following analysis towards its gold-plated side with little beam.
Fig. 5 shows is the situation of change that SiNx-Au micro-cantilever free end displacement z varies with temperature Δ T before the absorption PNIPAM molecule.This distortion is because SiN
xCause different with the thermal expansivity of Au.In the experimental temperature scope, have by linear elasticity small deformation theory: Δ z=3Kl
2Δ T.Wherein K is a constant, and l is a micro-cantilever length.Fig. 5 shows that micro-cantilever free-ended acoplanarity displacement changes delta z and temperature variation Δ T are linear.After the gold-plated surface of little beam had adsorbed the PNIPAM molecule, the experimental measurements of little beam distortion that temperature variation causes as shown in Figure 6.The thermomechanical effect of this above-mentioned pair of material that wherein except the influence that the PNIPAM molecular conformation changes, also be coupled.Change caused little beam distortion (Fig. 7) with having adsorbed the deformation curve that the deformation curve of beam after the PNIPAM molecule deducts the absorption front-axle beam, only just can having drawn by the PNIPAM molecular conformation.Can calculate the surface stress variation of little beam by this deformation gauge, relation between they the two by Stoney ' s formula (G.G.Stoney.The tension of metallic films deposited by electrolysis[J] .Proc.R.Soc.London.Ser.A, 1909,82:172-175) provide:
Wherein R is the radius-of-curvature of little beam deformed, E, and v, t are the Young modulus of little beam, Poisson ratio and thickness, Δ σ
1, Δ σ
2The surface stress changing value of corresponding little beam upper and lower surface.Be expressed as relation with the free-ended transversal displacement Δ of beam z:
L is little beam length.For that side of not adsorbing the PNIPAM molecule on little beam, its surface state remains unchanged in whole process.(Δ σ
1-Δ σ
2) in fact reflection is exactly the surface stress changing value that is adsorbed with that side surface of PNIPAM molecule.Be simply referred to as surface stress, result of calculation as shown in Figure 8.
Analyze in the aqueous solution the temperature variant folding process of PNIPAM molecule (Giuseppe Graziano.On the temperature-induced coil to globule transition of poly-N-isopropylacrylamide in dilute aqueous solutions [J] .Journal of BiologicalMacromoleculers freely, 2000,27:89-97): with lower critical solution temperature (LCST) is the boundary, the PNIPAM molecule presents hydrophobicity when being higher than this temperature, be the contact area of minimizing with hydrone, the PNIPAM strand is folding to be shunk, and forms the coccoid conformation of collapsing at last.When temperature was lower than LCST, the PNIPAM molecule presented water wettability, promptly wanted and water molecules, and the PNIPAM molecular stretching comes, and enlarged the contact area of self and hydrone, became the random coil shape conformation of swelling.This conformation transition from random coil to the bead of collapsing is finished than (1 ℃～2 ℃) in the close limit near LCST in the free solution.When PNIPAM molecule one end is restricted on the solid surface when forming molecular brush, carrying out (the E.B.Zhulina that this conformation transition of theoretical prediction is will be in the temperature range of broad continuous, O.V.Borisov, V.A.Pryamitsyn, T.M.Birshtein.Coil-globule type transitions in polymers.1.Collapse of layers of graftedpolymer chains[J] .Macromolecules, 1991; 24 (1): the 140-149) research of .SPR (Balamurugan S, Mendez S.Thermal Response of Poly (N-isopropylacrylamide) Brushes Probed by Surface Plasmon Resonance[J] .Langmuir, 2003, continuous the reducing of thickness of PNIPAM molecular brush 19:2545-2549) showing from 20 ℃ to 40 ℃, the experiment of QCM (Guangming Liu, Guangzhao Zhang.Collapse and swelling of thermallysensitive poly (N-isopropylacrylamide) brushes monitored with a quartzcrystal microbalance[J] .J.Phys.Chem.B 2005,109:743-747) show the water cut minimizing continuous in the PNIPAM molecular brush with the rising of temperature.
In this experiment, deducted the double material effect of little beam itself after, the just result of PNIPAM molecular brush influence just of its distortion performance is promptly to the reflection of PNIPAM molecule conformation transition process in the temperature change process.By shown in Figure 7, the distortion of beam continuous generation in 20 ℃～40 ℃, the conformation that this correspondence the PNIPAM molecule also is that continually varying---the result who mentions with the preceding paragraph is consistent at this therebetween.When temperature since 20 ℃ of risings, PNIPAM molecule in the molecular brush is changed to hydrophobicity gradually by water wettability, and (a) begin slowly to shrink among Fig. 9, distance reduces the strand that original repulsion mutually trails between the segment, also might run through between the adjacent PNIPAM molecule, tangle (b among Fig. 9).This contraction is equivalent to a kind of surface stress that the surface is tightened up concerning little beam, cause little beam to occur bending and deformation towards plating Au one side (being PNIPAM molecular brush one side), up to the intensification terminal point.At temperature-fall period, process has just taken place on the contrary, and promptly the PNIPAM strand is along with the reduction of temperature extends, the flexural deformation in the opposite direction of little beam.
Shown in Fig. 7 and 8, when temperature was reduced to 20 ℃, the distortion of beam still failed to recover fully, remaining distortion is promptly arranged or say that residual surface stress exists.With regard to whole process, exist tangible hesitation, be a kind of irreversible process.At free solution (Xiaohui Wang, Xingping Qiu, Chi Wu.Comparisonof the Coil-to-Globule and the Globule-to-Coil transition of a singlePoly (N-isopropylacrylamide) homopolymer chain in water[J] .Macromolecules, 1998; 31:2972-2976) and at QCM (Guangming Liu, GuangzhaoZhang.Collapse and swelling of thermally sensitive poly (N-isopropylacrylamide) brushes monitored with a quartz crystalmicrobalance[J] .J.Phys.Chem.B 2005,109:743-747), SPR (Balamurugan S, Mendez S.Thermal Response of Poly (N-isopropylacryl amide) Brushes Probedby Surface Plasmon Resonance[J] .Langmuir, 2003, also observed similar phenomenon in experimental study 19:2545-2549).This is because when the PNIPAM molecule is collapsed into the bead conformation, formed intramolecular interaction force (being commonly referred to be hydrogen bond) in the bead, this stronger effect continues keeping the collapsed state of bead when cooling, the PNIPAN molecule just can overcome it and extend when lower temperature, so the distortion during cooling always when heating up big (sluggishness) on same temperature spot.On the other hand, contingent entanglement between the PNIPAM strand in the process of intensification needs the long time to untie in temperature-fall period, and this also means the sluggishness that little beam is out of shape simultaneously in temperature-fall period.Fig. 9 has provided the synoptic diagram of whole conformation transition process.The conformation change that Fig. 4 has provided the PNIPAM molecule causes little beam distortion synoptic diagram.
The present invention proposes a kind of new method based on micro-cantilever Research on Sensing molecular folding/conformation change.The transformation of PNIPAM molecular conformation with temperature observed in distortion when varying with temperature by detecting the micro-cantilever that is adsorbed with the PNIPAM molecule on the single side surface, finds the continuous generation in 20 ℃～40 ℃ scopes of this transition process.Whole process is irreversible and has tangible hysteresis to answer.On the other hand, the variation of micro-cantilever surface stress, reflection be the information of intermolecular interaction, this microscopic mechanism to the recognizing molecule conformation transition is highly significant.
Embodiment 2, utilize micro-cantilever sensing experiment technology for detection lysozyme molecule conformation transition
Experimental system is as shown in Figure 3. semiconductor laser (6328nm, the laser beam of 2mW) sending focuses on the free end of little outstanding three arm beams. and folded light beam enters the detection target surface of photoelectric position sensor (PSD). and little beam is fixed in the small container of the stainless steel wall that glass cover binds (1ml), little beam length 200um, wide 20um, thick 1um. solution turnover is by the control of micrometeor peristaltic pump. by the electrothermal refrigeration device control solution temperature of container bottom, its temperature stability is ± 0.01K, experimental temperature is 20 ℃. when little beam occurs bending and deformation, laser beam is deflection thereupon, PSD is converted into corresponding electric signal output with this signal, can realize the detection to the beam distortion thus. and the minimum distinguishable displacement of little beam free end is 0.1nm. in the experimental system
In order to observe the transition phenomenon of lysozyme molecular conformation with temperature, earlier lysozyme to be modified on plating Au one side surface of the little beam of SiNx (Figure 10), concrete grammar is as follows: interact earlier 11mua (SH (CH by the Au-sulfydryl
2)
11COOH) be connected on the Au face, then by the amino (NH in the lysozyme
2) form amido link with the carboxyl (COOH) of 11mua and lysozyme is connected with 11mua, thereby finish the modification of lysozyme.The another side of little beam can not react in the whole process.In below analyzing with the forward that be bent into distortion of little beam towards its gold-plated side.
Character by solvent around changing (from phosphate solution PBS to hydrochloric acid croak solution) is observed the transformation of lysozyme conformation: to be in pH be that little beam is in steady state (SS) (flow velocity 0.25ml/min) this moment 7.4 the PBS solution to little beam during beginning.Implantation concentration is the hydrochloric acid croak solution of 6M during 600s.Because can interacting with the lysozyme peptide chain, the croak group in the hydrochloric acid croak make the hydrophobic grouping that included originally in the lysozyme expose, finally caused the sex change of lysozyme, promptly changed loose ball of string shape conformation (unfolding attitude) into from original ellipsoid shape nature conformation (folded state).As can be seen from Figure 11, flow into the hydrochloric acid croak after displacement rise rapidly, this deformation process promptly corresponding the folding deformation process of lysozyme conformation.Through reaching a stabilised platform after about 1000s, this explanation lysozyme sex change completely at this moment (unfolding).After stablizing 600s, will flow stops, and displacement begins rapid whereabouts, through slowing down gradually behind the 2500s, fall slowly afterwards always, this variation may be since solution flow/stop to cause.Be the hydrochloric acid croak solution of 6M once more with the 0.25ml/min implantation concentration when about 12500s, displacement is risen once more rapidly.After reaching a stable platform flow velocity is reduced to zero gradually from 0.25ml/min.The step that shows among Figure 11 is exactly this reaction of landing gradually.Injection pH is 7.4 PBS solution when 19300s, and displacement is earlier upwards then fallen rapidly.Displacement upwards is owing to flow velocity causes, and afterwards rapid corresponding the recovery of lysozyme conformation when hydrochloric acid croak solution is got back to PBS of falling, just the lysozyme conformation is from the performance of the conformation transition of unfolding attitude recovery folded state.Whole process is consistent with existing result of study: when lysozyme is among the PBS, present ellipsoid shape conformation; And PBS is changed into after the hydrochloric acid croak, presenting a ball of string that scatters, conformation is recovered when replacing back PBS.Mechanical signal from this conformation transition process, this experimental result show when the little beam that is adsorbed with lysozyme is among the PBS, and axiolitic conformation makes mutual repulsion between the molecule.When solution was changed to the hydrochloric acid croak, axiolitic conformation was scattered.This weakens intermolecular phase repulsive interaction, makes the flexural deformation of micro-cantilever generation forward.And when with solution when the hydrochloric acid croak changes PBS into again, the conformation of lysozyme is recovered the ellipsoid shape.Beam has produced the flexural deformation of negative sense like this.
Claims (7)
1. the method for a monitoring molecule conformation transition, be with molecular adsorption on the micro-cantilever single side surface, detect the micro-cantilever change in displacement that causes by molecule, the micro-cantilever displacement that is caused by molecule changes as described, then the conformation of described molecule changes; The thickness of described micro-cantilever is 0.1-2 μ m, and long is 40-1000 μ m, the wide 20 μ m that are at least.
2. method according to claim 1, it is characterized in that: the described micro-cantilever displacement that is caused by molecule conformation transition is: causing under the same environmental conditions of conformation transition that the micro-cantilever displacement of having adsorbed described molecule is poor with the micro-cantilever displacement of not adsorbing described molecule.
3. method according to claim 2 is characterized in that: described environmental baseline comprises temperature, pressure, irradiate light, adds the electromagnetic field change, organic solvent and pH value and ion concentration.
4. method according to claim 3 is characterized in that: described organic solvent is urea or guanidine.
5. according to claim 1 or 2 or 3 or 4 described methods, it is characterized in that: described molecule comprises synthetic macromolecule and natural macromolecular.
6. method according to claim 5 is characterized in that: described synthetic macromolecule is a poly-N-isopropyl acrylamide.
7. method according to claim 5 is characterized in that: described natural macromolecular is a lysozyme.
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| US20050048539A1 (en) * | 2003-06-13 | 2005-03-03 | The General Hospital Corporation | Methods to monitor molecule conformation and molecule/molecule proximity |
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